Fructose-1,6-diphosphate (hereinafter FDP) is a metabolic intermediate in the glycolytic pathway that is of graining importance in the field of medicines (Magalini S. I., Bondoli A., Scrascia E., Resuscitation, 5, 103 (1977) and Markov A. K., Oglethorpe N. C., Blake T. M., Lehan P. H., Hellems H. K., Am. Heart J., 100, 639 (1980)). To meet this increase need for fructose-1,6-diphosphate, an industrial process for its production is desired.
At present, FDP is produced commercially using yeast mediated phosphorylation. This process uses yeast and a nutrient mixture containing glucose and inorganic phosphoric acid. The yeast to increase the permeability of its cell wall has been pre-treated with toluene, for example, is able to synthesize FDP through Harden-Young type fermentation (Seizaburo YAMAGUCHI, Fermentation, ed by Iwanami Zensho, 134-143 (1953)).
In JP-A-62-272977 (corresponding to German Patent Publication No. 3,709,718) (the term "JP-A" as used herein means an unexamined published Japanese patent application), this process has been improved by using enzymes that are effectively immobilized in the yeast by a glutaraldehyde treatment that prevents the enzymes from permeating out through the yeast cell wall. In addition, JP-A-63-87993 (corresponding to German Patent Publication No. 3,726,182 and British Patent Publication No. 2,196,967) teaches using hollow-fiber ultrafiltration to separate permeable substances from the yeast to allow continuous production of fructose-1,6-diphosphate. These are the only methods for the production of FDP used commercially at the moment.
A method as yet undeveloped to produce FDP in which a FDP synthesizing enzyme is reacted with a substrate in vitro is also possible. In this process, for example, using glucose as a substrate, FDP could be synthesized through the three step reaction shown below: ##STR1## where the meaning of the symbols are as follows: Glc: glucose, G6P: glucose-6-phosphate, F6P: fructose-6-phosphate,
FDP: fructose-1,6-diphosphate, PA0 HX: hexokinase, PGI: phosphoglucose isomerase, PA0 PFK: phosphofructokinase, ATP: adenosine-5'-triphosphate, PA0 ADP: adenosine-5'-diphosphate.
In the above reaction, ATP must be continuously supplied to produce FDP because the ATP is consumed in both the first step and the third step of the reaction. This need for ATP, which is relatively expensive, prevents the use of this process for the commercial production of FDP.
Recently, an enzyme system has been developed which regenerates ATP in vitro from AMP (adenosine-5'-monophosphate) or ADP. Such an ATP-regeneration system is expected to allow ATP to be recycled, thus reducing its cost. This would enable industrial production of various useful substances using ATP. For example, by employing an adenylate kinase and an acetate kinase to convert AMP to ATP, with acetyl phosphate as a phosphate donor, a synthetic reaction system can be designed to produce physiologically active substances such as acetyl CoA, asparagine, pantothenic acid, and guanylic acid. See, JP-A-59-106296 (corresponding to Canadian Patent No. 1,194,825 and EP-A-84975). JP-A-54-122793 teaches a method in which ATP is supplied from an ATP-regeneration system employing acetate kinase, to produce glutathione from glutamic acid, cysteine, and glycine under the action of .gamma.-glutamylcysteine synthetase.
The above-described methods utilizing the phosphorylating ability of yeast are necessarily accompanied by alcohol fermentation and the generation of ethanol and carbon dioxide in large quantities as by-products. As a result, the amount of FDP actually produced from the conversion of glucose is low. In addition, the separation of FDP from ethanol and carbon dioxide is extremely troublesome. Carbon dioxide is also a serious problem where a column reactor packed with yeast is employed. Yet another difficulty is the necessity, and expense of adding ATP or AND (nicotinamide adenine dinucleotide) as energy sources for the fermentation reaction
It appears that a solution to the above problems would be an effective enzymatic process coupled with an ATP-regenerating system that does not give ethanol and carbon dioxide as by-products, and achieves a high conversion efficiency. Such a system would require the use of purified enzymes for the ATP-regeneration system and the physiologically-active-substance-synthesizing system. The purification of such enzymes takes considerable time and involves significant labor despite recent progress in the separation and purification of natural high-molecular substances such as enzymes. Accordingly, such enzymatic processes have not been economically feasible.
As mentioned above, current methods for producing fructose-1,6-diphosphate all involve such problems as the generation of unwanted by-products, a low conversion efficiency, and the necessity of extensive enzyme purification.